Method for immobilizing biologic molecules on solid surfaces

ABSTRACT

The invention provides a method for immobilization of biological molecules such as nucleic acids, peptides and proteins onto the surface of a glass or plastic solid support.

The present invention relates to a method to attach biologicalmolecules, such as oligonucleotides, peptides and proteins onto thesurface of a glass or plastic support. The immobilization of biologicalmolecules onto different substrates plays a crucial role in thedevelopment of the DNA microarray technology (Nature Genetics SupplementVol 21, 1999). In recent years, the DNA microarray technology is gainingincreasing acceptance in different areas of biomedical analysis. Thetechnique, thanks to its versatility and miniaturization, has determineda considerable advancement in the sensitivity and throughput ofdifferent analysis. The coating methods used in the production ofmicroarray slides represent a key factor for the success of thetechnique. Over the last few years, polymeric coatings have beendeveloped based on polyacrylamide or polydimetylacrylamide gels forregioselective immobilization by the 3′ or 5′ end of oligonucleotides. Aprocedure for immobilizing DNA in polyacrylamide and dimethylacrylamidegels was developed by the Mirzabekov group at the Engelhardt Instituteof Moscow (U.S. Pat. No. 0,981,734). The method consists of introducingfunctional groups onto a suitable polymeric support.

U.S. Pat. No. 5,861,247 discloses a method for constructingoligonucleotide matrices, which comprises confining a light sensitivefluid to a surface, exposing said light-sensitive fluid to a lightpattern so as to cause the fluid exposed to the light to coalesce intodiscrete units and adhere to the surface and contacting each of theunits with a set of different oligonucleotide molecules so as to allowthe molecules to disperse into the units. The procedure to fix a regularset of polyacrylamide gel pads on a glass slide is cumbersome as itinvolves silanization of the glass and photopolymerization ofacrylamide.

In another approach, Boles and coworkers at the Mosaic Technologies Inc.(U.S. Pat. No. 5,932,711 and U.S. Pat. No. 6,180,770) have developedchemistry for solid phase attachment of oligonucleotides based on thesynthesis of oligonucleotides bearing 5′-terminal acrylamidemodifications. Oligonucleotides bearing polymerizable functions arecopolymerized with acrylamide/bisacrylamide and covalently attached toan organosilane surface to which acryloyl groups have previously beengrafted. Also this procedure is time consuming and requires a carefulcontrol of operative parameters.

U.S. Pat. No. 6,121,027 discloses a process for the production of poly-,difunctional reagents having a polymeric backbone, one or more pendentphotoreactive moieties, and two or more pendent bioactive groups. Thereagent can be activated to form a bulk material or can be brought intocontact with the surface of a previously formed biomaterial andactivated to form a coating. The pendent bioactive groups function bypromoting the attachment of specific nucleic acids and other moleculesthat are capable of binding noncovalently to specific and complementaryportions of molecules.

U.S. Pat. No. 5,858,653 discloses a method to produce a polymericsupport for oligonucleotide and DNA attachment. In this patent, avariety of homopolymers and copolymers resulting from addition orcondensation polymerization provide a polymeric backbone capable ofbearing ionic groups, photogroups and thermodynamically reactive groups.The method is based on the activation of latent photoreactive groups togenerate active species which undergo addition to other active specieson the same or on another molecule in such a way that a tridimensionalnetwork is generated in which reactive functions are properly spacedfrom the surface. Although very flexible, also this method involves thesynthesis of complex copolymers whose composition may be difficult to becontrolled.

Object of the present invention is a simple and fast method forimmobilizing biological molecules such as peptides, proteins and nucleicacids onto the surface of glass or plastic materials commonly used assubstrates for the adhesion of said molecules, such as microwell plates,beads, tubes, microscope slides, silicon wafers and membranes. Suitableplastic materials are, for instance, made of polystyrene, polycarbonate,polyvinylchloride and polypropylene. The method of the inventionexploits the ability of some copolymers of N-substituted polyacrylamidesto be adsorbed onto the surface of the above mentioned materials, and,in some cases, once absorbed, to covalently react with the surfacethrough appropriate functional groups, forming a highly hydrophiliccoating with accessible functionalities. The coating bears reactivegroups able to covalently bind the biological molecules of interest.

The polymers object of the present invention are obtained throughradical polymerization of a mixture comprising

-   -   a) a monomer selected from the group comprising acrylamide,        methacrylamide, preferably mono- or di-substituted on the        nitrogen by C1-C12 liner or branched alkyl chains, which in turn        can be substituted by a halogen, preferably fluorine, or by a        methoxy group or, in the case of N-mono substituted compounds,        by a hydroxy group;    -   b) an ethylene or acrylic monomer linked by means of C1-C6 alkyl        chains optionally interrupted by O, N or S atoms, to functional        groups able to covalently react with amines, thiols or hydroxyls        present on a protein/peptide, or with the amino-modified residue        of a nucleic acid, wherein said functional groups are preferably        succinimide, oxiranes, carboxylic acids;

and, optionally,

-   -   c) an ethylene monomer bearing groups able to covalently react        with the glass silanols, preferably epoxy functions,    -   d) an ethylene monomer carrying an ionizable group which assumes        a positive charge in aqueous solution, preferably ammonium        groups.        The content of monomer (b) in the polymerization mixture is from        0.1 to 25% w/v, preferably 1-4%, the remaining part being        constituted by monomer (a). The content of monomers (c) and (d)        can be up to 25% w/v, preferably 1-4% for monomer (c) and 5-10%        for monomer (d). Monomer (d) cooperates with monomer (c) to        increase the adhesion of the polymeric coating and thus of        biological molecules to the support by combining covalent and        electrostatic interactions.

Examples of groups able to covalently bind biological molecules arecarboxylic acids active esters, optionally substituted 4- or 5-memberedcyclic carboxyimides, such as optionally substituted succinimide andmaleimide and oxirane. Such groups can be already present in one or moremonomer of the starting polymerization mixture or they can be introducedafter polymerization (Timofeev et al., Nucleic Acid Research, 1996, 24,16, 3142-3148). Examples of groups able to electrostatically interactwith biological molecules are amino and quaternary ammonium groups.

Preferred monomers according to the invention are N,N-dimethylacrylamide (monomer (a)); allyl oxyalkyl(C1-C4)oxiranes, N-acryloyloxysuccinimide or N-acryloyloxyalkyl(C1-C4)succinimide, and acryloylcarboxy acids in which the carboxy group is spaced from the acryloylresidue by a C1-C5 alkyl chain (monomers (b)), allyl oxymethyl oxiraneand N-acryloyloxysuccinimide being particularly preferred; glycidylmethacrylate (methacrylic acid 2,3-epoxypropyl ester) and allyl glycidylether (allyl 2,3-epoxypropyl ether) (monomers (c)); N,N,N,-trimethylaminoethylacrylamide (monomer (d)).

Preferred polymers are obtained from the following monomeric mixtures:

-   -   a) N,N-dimethylacrylamide and N-acryloyloxysuccinimide        ((a)+(b));    -   b) N,N-dimethylacrylamide, N-acryloyloxysuccinimide and        N,N,N-trimethylaminoethylacrylamide ((a)+(b)+(d));    -   c) N,N-dimethylacrylamide, acrylic acid, glycidylmethacrylate        ((a)+(b)+(c)).

The polymerization reaction can be carried out in apolar organicsolvents, preferably tetrahydrofuran, and is usually catalyzed byradicalic catalizers, such as α,α′-azoisobutyronitrile (AIBN). Whenusing polymers such as N,N-dimethylacrylamide, glycidyl methacrylate,acrylic acid ((a)+(b)+(c)), the polymerization reaction is carried outin water using ammonium persulfate and tetraethylenemethylenediamine(TEMED) as catalysts. According to the invention, the above mentionedpolymers are adsorbed on the substrate surface by contacting an aqueoussolution containing them with said surface for a time that can varydepending on the mixture used and the surface to treat, but that willusually range from a few seconds to 30 min and more, so as to form ahighly hydrophilic coating in which the reactive functions or groups areaccessible to the biological molecules. Afterwards, in case the monomerable to bind the biological molecules has to be activated in situ,either the acid will be transformed into the reactive ester or theprotein molecule or modified DNA will be directly coupled in thepresence of a dehydrating agent such as dicyclohexylcarbodiimide ordiisopropylcarbodiimide. When the monomer is already activated in thepolymer, before adsorption, proteins and modified DNA will be directlylinked covalently to said reactive groups, according to known procedures(DNA Microarrays A Practical Approach, Mark Schena Ed., OxfordUniversity Press). The aqueous solutions used for the deposition of thepolymeric coating have a polymer content ranging from 0.1 to 20% w/v,preferably from 0.1. to 1% w/v. In a preferred embodiment, the aqueoussolution contains 20% saturation ammonium sulfate.

The affinity for the substrate is such that the polymers adsorptiongenerates a coating which cannot be removed from the substrate surfaceby the usual buffers, even in the presence of additives such as urea,SDS, salts or at high temperature. In particular, polymers containingepoxy groups are attached to the glass by a mixed adsorption/covalentmechanism. The presence of covalent binding sites further stabilizes thecoating.

According to a preferred embodiment of the invention, polymers are usedfor coating DNA microarrays, peptides or proteins, which can be used inhybridization techniques with complementary molecules. Examples ofcomplementary moleculesare antigen/antibody, ligand/receptor,enzyme/substrate, protein/protein, preferably nucleic acid moleculesthat can be used in hybridization techniques according to wellestablished procedures. The invention also comprises substrates ofplastic or glassy materials, such as microwell plates, beads, tubes,microscope slides, silicon wafers and membranes, coated with thepolymers herein described.

DESCRIPTION OF THE FIGURES

FIG. 1: Fluorescence signals of hybridized oligonucleotides as afunction of the polymer concentration used for the coating. One nL of5′-amino modified oligonucleotide, 50 μM (femtomol/nanoliter), wasspotted on a glass slides coated with ammonium sulfate solutions of(DMA98-co-NAS2) at concentration 0.2, 0.4, 0.6, 0.8 and 1% w/v. Thespotted oligonucleotides were hybridized according to the protocolreported in the example section with a target complementaryoligonucleotide labeled at the 3′ end with Cy5 for 2 hours at 65° C.After washing, the slides were scanned with a Virtek scanner, and theimages were analyzed using the Virtek ChipReader software. Each valuerepresents the average fluorescence intensity value of six spots givenin arbitrary units.

FIG. 2: Fluorescence intensity of hybridized oligonuclotides vs. solventfrom which the coating [DMA98-co-NAS2] at 1% w/v concentration isadsorbed onto the slides. One nL of a 5′amino modified oligonucleotide(20 mer) was spotted at 50 μM concentration on slides coated with[DMA98-co-NAS2] dissolved at 1% w/v concentration in water,tetrahydrofuran and ammonium sulfate (20% of saturation).Oligonucleotides, spotted on different slides, were hybridized accordingto the protocol reported in the experimental section with thecomplementary probes labeled at the 3′ end with Cy5 for 2 hours at 65°C. After washing, the slides were scanned with a Virtek scanner, and theimages were analyzed using the Virtek ChipReader software. Eachfuorescence intensity value represents an average value of 6measurements.

FIG. 3: Average fluorescence intensity of spots as a function of theamount of spotted oligonucleotide. One nL of a 1, 5, 25 and 50 μMsolution of an amino modified oligonucleotide (20 mer) was spotted onslides coated with [DMA98-co-NAS2] dissolved at 1% w/v concentration inammonium sulfate (20% of saturation). The spotted oligonucleotides werehybridized according to the protocol reported in the example sectionwith target complementary oligonucleotides labeled at the 3′ end withCy5 for 2 hours at 65° C. After washing, the slides were scanned with aVirtek scanner, and the images were analyzed using the Virtek ChipReadersoftware. Each florescence intensity value represents an average valueof 6 measurements and is given in arbitrary units.

FIG. 4: Oligo-oligo, hybridization experiment: 1 nL of 5′ amino-modifiedoligonucleotide, at 10 μM (left line), 25 μM (central line) and 50 μM(right line) concentration, was spotted onto a slide coated with a 1%w/v solution of [DMA98-co-NAS2], dissolved in ammonium sulfate (20% ofsaturation) and hybridized with a complementary probe according to theprocedure described in the Examples, labeled with Cy5 at the 3′ end for2 hours at 65° C. After washing, the slides were scanned with a Virtekscanner, and the images were analyzed using the Virtek ChipReadersoftware.

FIG. 5: oligo-cDNA hybridization experiment

A 5′ amino-modified oligo corresponding to a fragment of neomycin genefrom plasmid pEGFP-N1, in concentration 3.125, 6.25, 12.5, 25 and 50 μM(from left to right) was spotted and hybridized, according to theprocedure described in the Examples, for 2 hours at 42° C., with thecDNA complementary fragment labeled at the 3′ end with Cy5. Afterwashing, the slides were scanned with a Virtek scanner, and the imageswere analyzed using the Virtek ChipReader software.

The following examples illustrate in detail the invention.

EXAMPLE 1

Synthesis of N-acryloyloxysuccinimide

To a solution of N-hydroxysuccinimide (NAS) (1.15 g, 10.0 mmol) andtriethylamine (1.53 ml) in chloroform (15 ml), acryloyl chloride (0.99g, 11.0 mmol), cooled at 0° C., was added dropwise, under mechanicalstirring, over a period of 30-min. After an additional stirring of 20min at 0° C., the solution was washed with ice-cold water (8 ml for 2times), dried on Na₂SO₄ and then filtered. 2,5-Di-tert-butylhydroquinone(0.5 mg) (polymerization inhibitor) was added to the chloroformsolution, which was concentrated to a volume of 3 ml, using a rotaryevaporator and filtered. Ethyl acetate (3 ml) and n-hexane (2 ml) wereslowly added while stirring to the chloroform solution, which was leftat 0° C. for several hours. The precipitate, a colorless solid, wasseparated by filtration and washed with an ice-cold solution of ethylacetate/n-hexane (4/1) and then washed only with n-hexane.

¹³C-NMR (DMSO), δ (ppm): 150 (carbonyl), 137 (CH₂═), 122 (—CH═), 24.8(—CH₂—)

EXAMPLE 2

Synthesis of poly (N,N-dimethylacrylamide-co-N-acryloyloxysuccinimide)[DMA98-co-NAS2].

In a 25 ml, round-bottomed flask, equipped with condenser, magneticstirring and nitrogen connection, N,N-dimethylacrylamide (600 mg, 6,15mmol), N-acryloylsuccinimide (20.7 mg, 0.12 mmol) were dissolved in 6 mlof dry tetrahydrofuran (THF). The solution was degassed by alternating anitrogen purge with a vacuum connection, over a 30 min period. Two mg ofα,α′-azoisobutyronitrile (AIBN) were added to the solution which wasthen warmed to 50° C., and left at this temperature under a slightlypositive nitrogen pressure for 24 hours. After the polymerization wascompleted, the solution was evaporated using a rotary evaporator, thewhite solid was dissolved in chloroform and precipitated by addingpetroleum benzin. The supernatant was discarded and the whole procedurerepeated 2 times. The polymer was dried under vacuum for 24 h at roomtemperature and stored at 4° C.

¹³C-NMR (DMSO), δ (ppm): 174.6 (backbone carbonyl), 166 (succinimidecarbonyl) 40-30 (metylene carbons). The degree of succinimide insertionwas determined from the ratio of the integrals of backbone andsuccinimide carbons and it was found to be 1.5%.

EXAMPLE 3

Synthesis of [DMA90-co-NAS10].

The synthetic pathway is the same as reported above, with the onlydifference being the ratio of DMA (600 mg, 6.15 mmol) to NAS (103.4 mg,0.62 mmol). ¹³C-NMR (DMSO), δ (ppm): 174.6 (backbone carbonyl), 166(succinimide carbonyl) 40-30 (metylene carbons). The degree ofsuccinimide insertion was determined from the ratio of the integrals ofbackbone and succinimide carbons and it was found to be 7%.

EXAMPLE 4

Synthesis of poly(N,N-dimethylacrylamide-co-N-acryloyloxysuccinimide-co-N,N,N-trimethylaminoethylacrylamide).

The synthetic path is the same as for [DMA98-co-NAS2]:N,N-dimethylacrylamide (600 mg, 6,15 mmols), N-acryloyloxysuccinimide(20.7 mg, 0.12 mmols) and N,N,N-trimethylaminoethylacrylamide (47 mg(0.3 mmols) in 6 ml of anhydrous tetrahydrofuran (THF).

EXAMPLE 5

Synthesis of poly(N,N-dimethylacrylamide-co-glycidilmethacrylate-co-acrylic acid)[DMA94-GMA2-AAc4]

In a 25 ml, round-bottomed flask, equipped with magnetic stirring andnitrogen connection, N,N-dimethylacrylamide (459 mg, 4.7 mmol),glycidilmethacrylate (14.2 mg, 0.10 mmol) and acrylic acid (14.6, 0.20mmol) dissolved in 8.1 ml of water. The solution was degassed byalternating a nitrogen purge with a vacuum connection, over a 30 minperiod. One mg/μL of TEMED and 1 mg/μL of APS (from a stock solution 40%w/v) were added to the solution which was left under a slightly positivenitrogen pressure for 90 min. The solution was diluted to a finalconcentration of 0.5% and diluted 1:1 with a solution of ammoniumsulfate at 40% of saturation immediately before use for coatingpreparation.

EXAMPLE 6

Assay of the active ester content of [DMAn-co-NASm], with n=98, m=2; andn=90, m=10, in aqueous solution.

N-hydroxysuccinimide showed no UV absorption at 260 nm, however,

under basic conditions, an absorption peak appeared at this wavelengthdue to the presence of the anionic species 1, λ max=260 nm, ε=9700 M⁻¹cm⁻¹

Therefore the appearance of 1 upon alkaline hydrolysis can be used toassess the amount of NAS incorporated into the polymers and freelyaccessible to the hydrolysis. The appearance of 1 was followedspectrophotometrically at 260 nm at 25° C. After the reaction wascompleted and the increase of the absorbance leveled off, the activeester concentration was calculated from the extintion coefficient of 1.

[DMA98-co-NAS2] and [DMA90-co-NAS10] contained respectively 90 and 400μmol of active N-hydroxysuccinimide ester groups/g of polymer indicatingthat the accessible NAS groups are ˜1% and 4%.

EXAMPLE 7

Assay of the active ester content of [DMAn-co-NASm], with n=98, m=2; andn=90, m=10, grafted onto the surface of test tube.

A test tube (6 cm high, 0.8 large) was coated with a solution of polymerbearing NAS groups. The determination of the NAS groups accessible tohydrolysis after adsorption of the polymer onto the surface was carriedout by recording the variation of absorbance at 260 nm of an ammoniasolution used to hydrolyze NAS groups on the inner surface of the testtube. Again, an increase in UV absorption at 260 nm was determined bythe production of 1 upon hydrolysis.

For [DMA98-co-NAS2], the number of active NAS/mm², was 29.0 pmol/mm².

EXAMPLE 8

Glass Slides Coating

Coating the glass slides requires two steps, a) surface pretreatment andb) adsorption of the polymer. In the first step the slides were washedwith 1 M NaOH for 30 min, with 1 M HCl for 30 min, with water and dried.In the second step, pretreated glass slides were immersed for 30 min ina solution of polymer from 0.2 to 1% w/v dissolved in a water solutionof ammonium sulfate at 20% of saturation. The slides were then washedextensively with water and dried in an oven at 60° C. Effect of polymerconcentration on the fluorescence intensity after hybridization wasinvestigated. FIG. 1 shows the fluorescence signal of hybridizedoligonucleotides as a function of the polymer concentration used duringthe adsorbing stage. A 1% w/v polymer concentration provided the bestresults. Addition of ammonium sulfate to the polymer solution alsoimproved the fluorescence signal, whereas dissolution of the polymer inan organic solvent dramatically reduced the amount of polymer adsorbedonto the surface. FIG. 2 summarize the results in terms of fluorescencesignal obtained with polymer dissolved in different media.

EXAMPLE 9

Activation of slides coated with [DMA94-GMA2-AAc4] bearing a carboxylicacid as a precursor. Glass slides were coated with 0.025% polymersolution in ammonium sulfate as reported in example 8. After coating,the slides were dried under vacuum at 80° C. and subsequently immersedin a solution containing N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide,acetic acid and dimethylaminopyridine. The slides were whased with waterand immersed in a solution of N-hydroxysuccinimide, rinsed with waterand dried in a vacuum oven at 80° C.

EXAMPLE 10

Immobilization of Probes

DNA deposition: custom synthesized 3′-amine-modified olionucleotides andPCR products, previously desalted, were dissolved in 150 mM sodiumphosphate buffer at pH 8.5. Stock solutions 100 μM or 0.1-0.5 mg/mL wererespectively used for oligonucleotieds and PCR. DNA solutions werediluted to 25, 10 and 5 μM and spots of 1 nL were printed on coatedslides to form microarrays. FIG. 3 shows the fluorescence intensity ofthe hybrids as a function of the concentration of oligonucleotidesspotted onto the surface

Printed slides were placed in a storage box and the uncovered storagebox was placed in a sealed chamber, saturated with NaCl, and allowed toincubate at room temperature. Overnight incubation showed the bestresults, the minimum incubation time was 4 hours.

Hybridization protocol: the residual reactive groups were blocked byimmerging the printed slides in 50 mM solution of ethanolamine in 0.1 MTris, pH 9.0, containing 0.1% sodiumdodecilsulfate (SDS) at 50° C. for15 min. After discarding the blocking solution, the slides were rinsedtwo times with water and shacked for 15 to 60 min with 4× SSC/0.1% SDSbuffer, pre-warmed to 50°. After a brief rinse with water the slideswere treated in different ways depending on the nature of the probes. Inthe case of oligonucleotide arrays the slides were placed in the rackand centrifuged at 800 rpm for 3 min. In the case of double stranded DNAarrays, the slides were placed in boiling water for two minutes, rinsedtwice with water and centrifuged at 800 rpm for 3 min. Next, the targetmolecules (2.5 μL per cm²), were dissolved in an appropriatehybridization buffer, heated in a boiling water bath for two minutes,cooled and immediately applied to micrarrays prepared as describedabove. The slides, placed in a hybridization chamber were transferred toa humidified incubator at the appropriate temperature for 4-16 hours.

Wash and scan: The slides were washed with 2× SSC/0.1% SDS athybridization temperature for 5 minutes. This operation was repeated twotimes and was followed by two washing steps with 0.2× SSC and 0.1× SSC.The slides were dried and scanned. FIG. 4 shows the results of ahybridization experiment between oligonucleotides deposited on thesurface and oligonucleotides labeled with Cy5 and compares the resultswith those obtained with a commercial slides under identical conditions.

1. A polymer for the immobilization of biological molecules onto a solidsupport, obtainable by copolymerization of a mixture comprising: a) amonomer selected from acrylamide and methacrylamide, optionally mono-ordi-substituted on the nitrogen by C1-C12 linear or branched alkyl chainswhich in turn can be substituted by a halogen, preferably fluorine, orby a methoxy group or, in the case of N-mono substituted compounds, by ahydroxy group; b) an ethylene or acrylic monomer linked by means ofC1-C6 alkyl chains optionally interrupted by O, N or S atoms, tofunctional groups able to covalently react with amines, thiols orhydroxyls present on a protein/peptide, or with the amino-modifiedresidue of a nucleic acid.
 2. A polymer according to claim 1, obtainableby polymerization of a mixture further containing: c) an ethylenemonomer bearing groups able to covalently react with the glass silanols,preferably epoxy groups, d) an ethylene monomer carrying an ionizablegroup which carries a positive charge in aqueous solution.
 3. A polymeraccording to claim 1, wherein the functional groups of monomers (b) arecarboxyimides, carboxylic acids or esters, oxirane.
 4. A polymeraccording to claim 3, wherein said functional group is succinimide ormaleimide.
 5. A polymer according to claim 1, wherein monomers (c)contain epoxy groups.
 6. A polymer according to claim 1, whereinmonomers (d) contain amine or ammonium groups.
 7. A polymer according toclaim 1, wherein monomer (a) is N,N-dimethylacrylamide.
 8. A polymeraccording to claim 1, wherein monomers (b) are allyl oxymethyl oxiraneor N-acryloyloxysuccinimide;
 9. A polymer according to claim 1, whereinmonomers (c) are glycidyl methacrylate or allyl glycidyl
 10. A polymeraccording to claim 1, wherein monomer (d) is N,N,N,-trimethylaminoethylacrylamide
 11. A polymer according to claim 1, obtained fromthe following monomeric mixtures: a) N,N-dimethylacrylamide andN-acryloyloxysuccinimide ((a)+(b); b) N,N-dimethylacrylamide,N-acryloyloxysuccinimide and N,N,N-trimethylaminoethylacrylamide((a)+(b)+(d)); c) N,N-dimethylacrylamide, acrylic acid,glycidylmethacrylate (a)+(b)+(c)).
 12. A method for coating a plastic orglass surface, which comprises contacting an aqueous solution of apolymer as claimed in claim 1 with said surface for a time sufficientfor polymer adsorption.
 13. A method according to claim 12, wherein theaqueous solution contains from 0.1 to 20% of said polymer.
 14. A methodas claimed in claim 12, wherein said aqueous solution contains 20%saturation ammonium sulfate.
 15. A method for the immobilization ofbiological molecules on the surface of a plastic or glass support whichcomprises coating said surface according to claim 12 and attachingthereto the selected biologic molecule.
 16. A method according to claim12, wherein said biologic molecules are peptides, proteins or DNA.
 17. Asolid support for biological molecules having at least one surfacecoated with a polymer according to claim
 1. 18. A solid supportaccording to claim 17, which is selected from multi-well plates, beads,tubes, microscope slides, wafers and silica membranes.
 19. Use of apolymer according to claim 1, or of a solid support according to claim17, for the immobilization of biological molecules.